Softworx imaging software

Images were acquired using a DeltaVision Elite wide-field fluorescence microscope (GE Healthcare). Live cell images were generated using a 60x/1.42 NA oil-immersion objective; fixed cell images were generated using a 100x/1.40 NA oil-immersion objective. Specific imaging conditions for each experiment are indicated in Supplementary file 4. Images were deconvolved using softWoRx imaging software (GE Healthcare). Unless otherwise noted, images were maximum intensity z-projected over the range of acquisition in FIJI (RRID:SCR_002285, Schindelin et al., 2012 (link)).

Cells for imaging experiments were grown to exponential phase in CSM + 2% dextrose. Cells were then transferred onto Concanavalin A-treated 96-well glass bottom Corning plates (Corning, Corning, NY). For experiments described in Figure 4A–C and Figure 4—figure supplement 1B–C cells were visualized at room temperature using the DeltaVision Elite Imaging System with softWoRx imaging software (GE Life Sciences, Marlborough, MA). The system was based on an Olympus 1 × 71 inverted microscope (Olympus, Japan), and cells were observed using a UPlanSApo 100 × 1.4 NA oil immersion objective. Single plane images were acquired using a DV Elite CMOS camera. For experiments described in Figure 4D–F and Figure 4—figure supplement 1D, cells were visualized at room temperature using an inverted epi-fluorescence microscope (Nikon Ti) equipped with a Spectra X LED light source and a Hamamatsu Flash 4.0 sCMOS camera using a 100x Plan-Apo objective NA 1.4 and the NIS Elements software. Quantification of co-localization was performed on all planes of a 3D stack using the Colocalization Threshold tool in FIJI. Image processing for PB counting was performed using Diatrack 3.5 particle tracking software.

Samples were grown overnight in synthetic media containing 2% glucose, diluted to OD₆₀₀ = 0.05 or 0.1 the following day, and grown to mid-log phase (OD₆₀₀= 0.3–0.8). Cells were harvested by centrifugation and washed in ¼ volume of fresh synthetic media +/− 2% glucose, then harvested again and resuspended in 1 volume of fresh synthetic media +/− 2% glucose and grown 15 min at 30°C. Cells were then transferred onto Concanavalin A-treated MatTek dishes (MatTek Corp., Ashland, MA) and visualized at room temperature using the DeltaVision Elite Imaging System with softWoRx imaging software (GE Life Sciences, Marlborough, MA). The system was based on an Olympus 1X71 inverted microscope (Olympus, Japan), and cells were observed using a UPlanSApo 100 × 1.4 NA oil immersion objective. Single plane images were acquired using a DV Elite CMOS camera. Image processing for PB analysis was performed using Diatrack 3.5 particle tracking software as described below.

For time course imaging of cells expressing GFP-IME1, SWI4-mCherry, or WHI5-mCherry, 500 µL of meiotic culture was fixed with a final concentration of 3.7% formaldehyde (v/v) at room temperature for 15  min. Cells were then washed in 1  ml of 100  mM potassium phosphate [pH 6.4] and stored at 4 °C in 20  µl of KPi Sorbitol solution overnight (100  mM potassium phosphate [pH 7.5], 1.2 M sorbitol). Cells were mounted on a slide and imaged using DeltaVision Elite wide-field fluorescence microscope (GE Healthcare) with a 60 x/1.516 oil immersion objective. Deconvolution of images was done with softWoRx imaging software (GE Life Sciences).
For live-cell imaging, cells at OD₆₀₀ of 1.85 in conditioned SPO (filter-sterilized SPO culture after ~5 hr in 30 °C) were sonicated and transferred to a concanavalin A (Sigma) treated 96-well clear, flat bottom plate (Corning). Four z positions (2 μm step size) were acquired per XY position. Acquisition was performed in a temperature-controlled chamber at 30 °C. Please refer to Supplementary file 7 for acquisition settings.